Knowledge of the molecular genetic basis for regulating hemoglobin synthesis is important both for a fuller understanding of human developmental biology and for new approaches to therapies for inherited anemias such as sickle cell disease or thalassemias. The distal locus control region (LCR) is a major regulator of mammalian beta-like globin genes, and is marked by 5 DNase hypersensitive sites (HSs). It functions by opening the beta-globin domain in chromatin and enhancing the expression of genes in that domain in a developmentally specific manner. Some models attribute the primary function of the LCR to short, discrete elements at the HSs. In contrast, a simultaneous alignment of beta-globin LCR DNA sequences from five mammalian species reveals a distributed pattern of conserved sequences, including many outside the minimal cores on the DNase hypersensitive sites (HSs) as well as several within the HS cores that have not been studied previously. Recent studies from the P.I.'s laboratory have shown that conserved E boxes within the core of HS2 are needed for full enhancement of globin gene expression, and that sequences located about 1 kb 5' to the HS3 core are needed both for an apparent domain opening activity in concert with the HS3 core and for the ability of rabbit DNA fragments containing HS3 to synergize with HS2. This latter region comprises a DNase HS in K562 cells and contains many conserved sequence blocks. The first two specific aims are designed to identify and characterize the proteins that function at the HS2 E boxes: (1) further functional tests of the HS2 E boxes and tests of the involvement of individual proteins in mediating the effects, and (2) identify other proteins that bind to the E boxes in HS2. This group of investigators has shown that the basic helix-loop-helix protein TAL1, which is required for hematopoiesis but for which no cis-acting target is known, binds to these E boxes in vitro, and the proposed experiments will test its possible role at the E boxes and other important hypotheses. The other four specific aims are designed to further our understanding of the structure and function of the region 5' the HS3 containing HS3.5: (3) higher resolution mapping of the nuclease HS3.5, (4) map the functional regions around human HS3.5 involved in establishment and maintenance of an active, open chromatin domain, (5) map the sequences in and around HS3 required for synergism with HS2, and (6) identify sequence-specific protein binding sites in HS3.5 and isolate the binding proteins.